Cell-specific targeting for delivery of effector moieties such as diagnostic or therapeutic agents is a widely researched field and has led to the development of non-invasive diagnostic and/or therapeutic medical applications. In particular in the field of nuclear medicine procedures and treatments, which employ radioactive materials emitting electromagnetic radiations as γ-rays or photons or particle emitting radiation, selective localization of these radioactive materials in targeted cells or tissues is required to achieve either high signal intensity for visualization of specific tissues, assessing a disease and/or monitoring effects of therapeutic treatments, or high radiation dose, for delivering adequate doses of ionizing radiation to a specified diseased site, without the risk of radiation injury in other e.g. healthy tissues. It is thus of crucial interest to determine and assess cell-specific structures and in particular structures that are present in case of tumors (i.e. cancer) or inflammatory and autoimmune diseases, such as receptors, antigens, haptens and the like which can be specifically targeted by the respective biological vehicles.
The folate receptor (FR) has been identified as one of these structures. The FR is a high-affinity (KD<10−9 M) membrane-associated protein. In normal tissues and organs FR-expression is highly restricted to only a few organs (e.g. kidney, lungs, choroids plexus, and placenta), where it largely occurs at the luminal surface of epithelial cells and is therefore not supplied with folate in the circulation. The FR-alpha is frequently overexpressed on a wide variety of specific cell types, such as epithelial tumours (e.g. ovarian, cervical, endometrial, breast, colorectal, kidney, lung, nasopharyngeal), whereas the FR-beta is frequently overexpressed in leukaemia cells (approx. % of acute myelogenous leukaemia (AML) are FR-beta positive). Both may therefore be used as a valuable tumour marker for selective tumour-targeting (Elnakat and Ratnam, Adv. Drug Deliv. Rev. 2004; 56:1067-84). In addition, the FR-beta isoform has been found on activated (but not resting) macrophages. Activated macrophages are involved in inflammatory pathologies such as e.g. rheumatoid arthritis, psoriasis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, atherosclerosis, diabetes, osteoarthritis, glomerulonephritis, infections, etc.
The literature reports several preclinical studies of folate-based imaging agents for detection/localization of sites of inflammation as well as folate receptor targeted therapy of these diseases. Recently, a clinical study has been published that reports the results of imaging studies in patients with rheumatoid arthritis using the FolateScan (Turk et al., Arthritis and Rheumatism 2002, 45, 1947-1955; Paulos et al., Adv. Drug Deliv. Rev. 2004, 56, 1205-1217; Chen et al., Arthritis Research & Therapy 2005, 7, 310-317; Hattori et al., Biol. & Pharm. Bull. 2006, 29, 1516-1520; Chandraseka et al., J. Biomed. Mat. Res. Part A 2007, 82, 92-103; Varghese et al., Mol. Pharmaceutics. 2007, 4, 679-685; Low et al. Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases 2008, 41, 120-129; Matteson et al., Clinical and Experimental Rheumatology 2009, 27, 253-259).
Folic acid, which is based on a pteridine skeleton conjugated through a benzoylamino moiety to a glutamate, and its derivatives have thus been intensively studied over the past 15 years as targeting agents for the delivery of therapeutic and/or diagnostic agents to cell populations bearing folate receptors in order to achieve a selective concentration of therapeutic and/or diagnostic agents in such cells relative to normal cells.
Various folic acid derivatives and conjugates are known and have been (pre)clinically evaluated, including folate radiopharmaceuticals (Leamon and Low, Drug Discov. Today 2001; 6:44-51; U.S. Pat. No. 4,276,280), fluorinated folate chemotherapeutics (U.S. Pat. No. 4,628,090), folate-conjugates with chemotherapeutic agents (Leamon and Reddy, Adv. Drug Deliv. Rev. 2004; 56:1127-41; Leamon et al, Bioconjugate Chem. 2005; 16:803-11), with proteins and protein toxins (Ward et al., J. Drug Target. 2000; 8:119-23; Leamon et al, J. Biol. Chem. 1993; 268:24847-54; Leamon and Low, J. Drug Target. 1994; 2:101-12), with antisense oliconucleotides (Li et al, Pharm. Res. 1998; 15:1540-45; Zhao and Lee, Adv. Drug Deliv. Rev. 2004; 56:1193-204), with liposomes (Lee and Low, Biochim. Biophys. Acta-Biomembr. 1995; 1233:134-44; Gabizon et al, Adv. Drug Deliv. Rev. 2004; 56:1177-92), with hapten molecules (Paulos et al, Adv. Drug Deliv. Rev. 2004; 56:1205-17), with MRI contrast agents (Konda et al, Magn. Reson. Mat. Phys. Biol. Med. 2001; 12:104-13) etc.
Folate radiopharmaceuticals can be in particular very useful for an improved diagnosis and evaluation of the effectiveness of cancer and inflammatory and autoimmune disease therapy. This may include assessment and/or prediction of a treatment response and consequently improvement of radiation dosimetry. Typical visualization techniques suitable for radioimaging are known in the art and include positron emission tomography (PET), planar or single photon emission computerized tomography (SPECT) imaging, gamma cameras, scintillation, and the like.
Both PET and SPECT use radiotracers to image, map and measure activities of target sites of choice. Yet while PET uses positron emitting nuclides which require a nearby cyclotron, SPECT uses single photon emitting nuclides which are available by generator systems, which may make its use more convenient. However SPECT provides less sensitivity than PET and beside a few approaches quantification methods are lacking. In case of PET, the positron annihilation results in two gamma rays of 511 keV which provide the basis for well developed quantification methods. Thus PET is one of the most sophisticated functional imaging technologies to assess regional uptake and affinity of ligands or metabolic substrates in brain and other organs and thus provides measures of imaging based on metabolic activity. This is for example achieved by administering a positron emitting isotope to a subject, and as it undergoes radioactive decay the gamma rays resulting from the positron/electron annihilation are detected by the PET scanner.
Factors that need to be considered in the selection of a suitable isotope useful for PET include sufficient half-life of the positron-emitting isotope to permit preparation of a diagnostic composition optionally in a pharmaceutically acceptable carrier prior to administration to the patent, and sufficient remaining half-life to yield sufficient activity to permit extra-corporeal measurement by a PET scan. Furthermore, a suitable isotope should have a sufficiently short half-life to limit patient exposure to unnecessary radiation. Typically, a suitable radiopharmaceutical for PET may be based on a metal isotope, such as gallium or copper. These two require however a chelator for entrapment of the metal, which may have an effect on steric and chemical properties. Alternatively a radiopharmaceutical may be based on a covalently linked isotope which provides minimal structural alteration. Radionuclides used for covalent attachment and which could be suitable for PET scanning are typically isotopes with short half lives such as 11C (ca. 20 min), 13N (ca. 10 min), 15O (ca. 2 min), 18F (ca. 110 min).
To date, a number of chelate-based folate radiopharmaceuticals have been synthesized and successfully evaluated as diagnostic agents for imaging folate receptor-positive tumors (e.g. with 111In, 99mTc and 67Ga (Leamon et al., Bioconjug Chem 2002, 13 (6):1200; Siegel et al., J. Nucl. Med. 2003, 44:700; Müller et al., J. Organomet. Chem. 2004, 689:4712; Müller et al. Bioconjug Chem 2008, 17(3):797; Müller et al. Nucl Med Biol 2011, 38 (5): 715) for SPECT or with 68Ga for PET (Mathias et al., Nucl. Med. Biol. 2003, 30(7):725; Fani et al., Eur J Nucl Med Mol Imaging 2011, 38 (1):108).
In addition, there is growing interest in folate radiopharmaceuticals having a covalently linked isotope, in particular a 18F-labeled folate radiopharmaceutical because of its excellent imaging characteristics, the long half-life of 18F (approximately 110 minutes) and because 18F decays by emitting positrons having the lowest positron energy, which allows for the sharpest images with a high-resolution PET. Furthermore, the longer half-life of 18F (compared to other isotopes such as 68Ga) also allows for syntheses that are more complex and satellite distribution to PET centers with no radiochemistry facilities.
To date, reports in the literature include 18F-labeled folic acid derivatives having the 18F isotope either directly linked to the folate molecule or through a prosthetic group (WO 2006/071754, WO 2008/098112, WO 2008/125613, WO 2008/125615, WO 2008/125617, Bettio et al., J. Nucl. Med., 2006, 47(7), 1153; Ross et al., Bioconjugate Chem., 2008, 19, 2402, Ross et al., J. Nucl. Med., 2010, 51(11), 1756).
Yet, many methodologies still suffer from drawbacks including time-consuming radiosyntheses giving low radiochemical yields, or unfavorable pharmacokinetics for molecular imaging purposes, and the like.
Thus, there is still a need for specific radiopharmaceuticals suitable for metabolic imaging of tumors to improve diagnosis and treatment of cancer and inflammatory and autoimmune diseases.
Applicants have now found efficient and versatile methods for production of new 18F-labeled folate radiopharmaceuticals wherein the 18F isotope is introduced via a prosthetic group, more specifically via a prosthetic group having a saccharide group, such as a cyclic mono- or oligosaccharide, which are preferably based on a pyranoside or furanoside. A prominent member of this group is e.g. 2-18F Fluoro-2-deoxy-D-glucose (18F-FDG), which is one of the most widely used PET tracer in the world for in vivo assessment of regional glucose metabolic rates in humans. Approved diagnostic uses with PET include its use for determination of myocardial viability and detection of cancer, epilepsy, and Alzheimer's disease. However, there are only very few examples using 18F-FDG as a building block or prosthetic group for the radiosynthesis of 18F-labeled compounds.
Applicants have found that the new compounds of the invention are able to overcome the drawbacks of known conjugates and meet the current needs by showing several advantages (due to e.g. their chemical and/or physical characteristics, specifically their hydrophilic character, etc.), such as improved labeling efficiency at low ligand concentration, better biodistribution, increased target tissue uptake and better clearance from non-targeted tissues and organs.
Moreover the new compounds of the invention are obtainable in good yields to meet the expectations for a clinical application in humans. In addition, the new radiosynthesis is applicable in an automated synthesis module which allows a fast and convenient labeling procedure which meets the requirements of GMP guidelines. Preliminary in-vitro and in-vivo studies suggested their suitability as powerful diagnostic agents for FR-positive tumours.